The complement system is ingeniously designed to prevent infections as well as to process immune complexes and damaged tissue. Strict control of its activation during innate and acquired humoral immune responses is critical to minimize damage to host tissue. Membrane cofactor protein (MCP; CD46) is widely expressed inhibitor of complement activation at the critical step of C3/C5 convertase generation. It serves as a cofactor for the serine protease factor I to cleave and thereby inactivate C3b and C4b that deposit on host tissue. Most cells and tissues express MCP as a family of four isoforms that differ in their O-glycosylation and cytoplasmic tails. MCP is a receptor for three human pathogens: measles virus, Streptococcus pyogenes and Neisseria. Attachment of the measles virus of Neisseria to MCP transmits signals for IL-12 down-regulation in monocytes or for calcium fluxes in epithelial cells, respectively. MCP has also been implicated in reproduction, in large part due to its dense expression on placental trophoblast and on the inner acrosome membrane of spermatozoa. Because of its potent complement regulator activity, MCP has been recombinantly produced for use as a soluble therapeutic agent and engineered into pigs whose organs are being employed for xenografting. In this grant application, we propose to continue our studies on the structure, microbial interactions and function of MCP. We postulated and later demonstrated during the prior grant period that each of four regularly expressed isoforms of MCP possesses functional advantages. In this renewal application we continue this focus while placing an increased emphasis on microbial connections and cell signaling. The active sites of MCP will be characterized by NMR spectroscopy and X-ray crystallography. We have recently demonstrated that in three cell types MCP is tyrosine phosphorylated on one of its two cytoplasmic tails. For this signaling event, we propose a systematic analysis of the site(s), responsible kinase(s), and related downstream events. Additionally, we will explore in depth the microbial interactions with MCP as they relate to binding sites, signaling events, and three-dimensional structure. We will characterize three strains of transgenic mice expressing human MCP. We anticipate these animals will be a valuable tissue source. We also propose a targeted disruption of the MCP mouse gene since MCP is expressed predominantly on the inner acrosomal membrane of mouse spermatozoa. The fertility of these mice will be assessed and they will be crossed with other deficient mice strains to explore the role of MCP in reproduction. Lastly, the mechanism of action of MCP in situ will be analyzed by quantitative methods developed during the prior granting period. We will address such questions as the role of membrane versus fluid phase inhibitors and contribution of MCP versus decay accelerating factor. The specific aims of this proposal outline a broad-based approach to increase our understanding of complement regulation by one if its major inhibitory proteins. Additionally, these experiments will expand our knowledge of the fascinating interactions of this complement regulator with three common infectious diseases and its role in cell signaling events and reproduction.